The purpose. To develop theoretical approaches concerning expediency of application of modern molecular-genetic methods of probes within the limits of holding genetic monitoring in system of preservation of genetic resources of animals. Methods. Dialectic method of scientific knowledge, analysis and systemic generalization. Results. It is necessary to consider ndigenous breeds as carrying agents of the unique genetic information which cannot be replicated by modern methods of selection. In some ecosystems economically expediently their growing in purity, and also straight introduction in zones where reproduction of highly productive forms is impossible. From this point of view, study of gene pools of indigenous breeds is important for searching new useful genes of quantitative or other attributes (productivity, resistance to diseases and stresses) which in the further can be used in selection. Conclusions. For comprehensive analysis of genetic structure of pedigree resources of breeds development and systematization of methods on determination of the most informative tests for different types of DNA-markers is necessary. Heading of elements of molecular-genetic monitoring will promote detection of carrying agents of valuable genes complexes by deriving molecular-genetic information of features of genetic structure. That will allow identifying and determining optimum content of gene pools and quality of genetic material for backfilling and keeping specimens of biomaterial.
| As a result of molecular-genetic analysis of two cow populations -Ukrainian Brown Dairy breed (UBD) and Sumy interbreed type of Ukrainian Black-and-White breed (SITUBWD) in the technique of ISSR analysis with the use of the fragment of microsatellite loci with trinucleotide core motif (AGC) 6 C (S1) as an amplification primer, there were identified 23 DNA-patterns. It was determined that genetic locus with the identified size of 1350 bp was monomorphic in animals of SITUBWD occurring in all study samples, however, in UBD breed cows the amplicons of this size were not detected at all (p<0.001). The population-genetic analysis of the two studied animal groups (taking into account maternal origin) by molecular-genetic testing in the ISSR-PCR technique revealed essential statistically significant differences. The total average number of PCR products obtained from the cows of the newly developed Sumy interbreed type of Ukrainian Black-and-White breed was 15,050 being 12,450 in Ukrainian Brown Dairy breed (p <0,001). A rather high level of genetic similarity between the animals of Ukrainian Brown Dairy and Sumy interbreed type of Ukrainian Black-and-White Dairy (0,768) is quite possibly connected with the influence of the unique aboriginal Lebedynsky breed on which maternal base they were developed. citation | Ladyka V, Skliarenko Y, Pavlenko Y, Metlytska O, Ivankova I (2019). Molecular-genetic analysis of cows genetic structure and determination of genealogical relatedness level of bulls of modern dairy breeds. Adv. Anim. Vet. Sci. 7(5): 405-411. doI | http://dx.
Assessment of swine leukocyte antigen polymorphism (SLA–3) in different breeds pigs of Ukraine was carried out using the method of allele-specific PCR. Features of the allelic fund structure in pig breeds such as Large white, Mirgorod, Ukrainian steppe white, Ukrainian steppe speckled and Vietnamese potbellied pig were studied on four polymorphic sites: SLA–3–0602, SLA–3–0401, SLA–30101 and SLA–3–03cs01. In the study population of Mirgorod pigs, the absence of SLA–3–03cs01 and SLA–3–0602 alleles was detected. The experimental animals were polymorphic by the SLA–3–0101 and SLA–3–0401 marker systems with the above alleles frequency of 18.2 and 81.8%, respectively. By the polymorphic site SLA–3–0401 pigs of Large white, Ukrainian steppe white and Vietnamese potbellied breeds were monomorphic, whereas the representatives of the aboriginal Ukrainian steppe speckled breed the frequency of this allele did not exceed 40%. The genetic structure of Vietnamese potbellied pigs significantly differed from the characteristics obtained for the local Ukrainian breeds by four SLA sites. The most statistically significant differences are recorded for allele frequencies SLA–3–0602 and SLA–3–0101. The SLA–3–0602 allele was absent in Vietnamese potbellied breed with its 100% presence in Large white pigs. In contrast, the SLA–3–0101 allele was found in all Vietnamese pigs and was absent in a sample of the Large white pigs (p < 0.05). The distribution pattern of alleles in all of the samples selected for the study of polymorphisms in the pig locus of leukocyte antigen (SLA–3) showed a high genetic homogeneity of the Large white animals sample. The SLA–3–0101 and SLA–3–03cs01 alleles carriers were not detected, and by SLA–3–0602 and SLA–3–0401, 100% of the specimens had the respective alleles in their genotype. High values of the genetic homogeneity in these animals are confirmed by calculation of the total heterozygosity index, which was 0.136. The maximum genetic and, accordingly, genealogical relationship (according to the summary zootechnical account) were recorded in Vietnamese potbellied pigs for which the index of intragroup similarity was 0.960, and the calculated heterozygosity was the least among the breeds under study – 0.024. It is expedient to consider the results of the population-genetic characteristics analysis of the autochthonous pig breeds – the Ukrainian steppe speckled and Mirgorod when further planning selection and breeding work with them. The minimum value of intragroup similarity was found in the group of the Ukrainian steppe speckled swine – 0.156, as compared to the values of this index in pigs of other breeds under study (p < 0.05), and the theoretically calculated heterozygosity for these animals sample was 0.823. The integral assessment of breeds genetic differentiation by SLA–3 polymorphisms was obtained by performing a cluster analysis based on the calculated values of genetic distances between the breeds. The most genetically distant ones were the Mirgorod and Ukrainian steppe speckled breeds, and the value of the genetic distance between them was 0.776. In general, Ukrainian steppe speckled was characterized by the most unique distribution of SLA–3 alleles compared to other breeds under study, which has influenced the configuration of the obtained dendrogram by the UPGMA method. By means of molecular genetic, population and cluster analysis, the singularity and specificity of the Ukrainian steppe white and speckled pigs are shown. The genetic characteristics of the Mirgorod breed obtained by us can be useful in the process of recreating these animals using biotechnological methods, since the only gene pool herd of these pigs was eliminated due to the African plague infection. The generative material from the Mirgorod breed pigs has only been preserved in the Bank of Genetic Resources of the M.V.Zubets Institute of Animal Breeding and Genetics, NAAS. Determining genetic characteristics of the SLA allelic fund breed specificity of the local pig breeds reveals the prospects for using this information as an additional tool for their genetic certification, in special programs for conservation and restoration of their gene pool.
Розроблена діагностична система скринінгу ендогенного ретровірусу свиней підтипу С (PERV-C) за допомогою мультиплексної ПЛР-SSP для виявлення особин із зниженим ризиком біологічної небезпеки при їх застосуванні для цілей ксенотрансплантації.На зразках ДНК, отриманих від тварин свиней порід в’єтнамський мейшан та велика біла, визначена чутливість та специфічність тест-системи PERV-C – α-Actin. Встановлено, що гранично допустимою концентрацію геномної ДНК для виявлення фрагменту ретровірусу свиней в ПЛР з наступним розділенням продуктів ампліфікації шляхом горизонтального електрофорезу в агарозному гелі є 15,2 пг/мкл, а мінімальна кількість ПЛР-продукту для можливості його візуалізації склала 5 × 103 копій.
Evaluation of livestock genomes to identify gene complexes which lead to expression of the quantitative trait desired for breeding is impossible without knowledge of its fine molecular structure and characteristics of "work" of this complex system – the interaction of genes at the level of their protein products, regulation of gene activity, and so on. To address such complex issues focused efforts of leading scientists in the world who have worked within the mapping and sequencing the genomes of the most economically important species of farm animals – "transcript" of the genome of some dog breeds was completed in 2003, the structure of the genome of cattle, pigs, rabbits, horses and turkeys was identified only in 2009, and the noticeabout completion of international program "Honeybee Genome" appeared only in 2010. The rapid genesis of methods of molecular genetic analysis of farm animals’ genomes is not in vain called "DNA Revolution" and the number of methodological approaches and developments in this field is huge, so we consider it appropriate to be limited to developments of a leading scientific center of Ukraine to address these important issues – Institute of Animal Breeding and Genetics nd. a. M.V.Zubets of NAAS of Ukraine. The main focus of the institute is developments of scientists which help to solve an important problem of the state –preservation of the existing gene pool of domestic breeds. In this sense, the developments of genetic testing of cattle on the "major" genes of quantitative traits, involved in forming the qualitative indicators of milk and meat productivity (k-Cn, βLG, GH, TG5, CAPN1 530, MSTN), have shown uniqueness of domestic breeds and high frequency of genotypes desired for breeding aimed at improving quality of milk and meat products. For example, k-Cn (kappa-casein) gene can be determined in a herd of animals with genotypes determining the presence of milk protein with desirable properties for cheese-making and selection of animals on some genotypes of βLG, GH, TG5 not only helps to intensify breeding towards milk yield increase, but will increase its fat content. Some mutation, identified in CAPN1 530 (calpain) gene, will provide for its carrier –a representative of beef cattle breeds by expressed marbling of muscle and individuals with abnormalities in MSTN (myostatin) gene will have "double" hypertrophied muscle structure. By the way, convenience to address practical issues of production of molecular genetic markers is that any genetic material (blood, pinched hair from an ear at marking of animals, buccal scrape of mucous membranes, urine, manure, semen, hair with hair follicles and even a few cells of embryos, etc.) can be used for researches at any age of animals. The value of this information is an early assessment of a genotype of an animal, that is, its genetic potential immediately after birth. Using crossbreeding of different local populations of farm animals with imported ones has helped to improve the genetic potential of productivity of many populations of animals in very short term and the widespread artificial insemination has created conditions for economically useful programmable transmission of genes from parents to offspring. But now, as a result of intensive breeding and breed formation the accumulated reserve of variability has been reduced that can’t affect the possibilityofstrategic breeding. Intensive processes of "holsteinisation" and use of the limited number of sires of leading lines of this breed at moderate inbreeding and limitation of effective population size have led to the accumulation of "genetic load" in herds of local breeds–mutant variants of gene with lethal and semi-lethal effects. Determination of hidden genetic abnormalities in cattle (BLAD – Bovine Leukocyte Adhesion Deficiency, CVM – Complex Vertebral Malformation, DUMPS – Deficiency of Uridine Monophosphate Synthase) using DNA diagnostics is carried out to prevent the accumulation of genetic load and reproductive losses. The vast number of spontaneous abortions and reasonsof early death of calves with birth defects is caused by gene mutations appearing phenotypically only if carriers of mutant alleles are both parents of a descendant. Identification of animals with heterozygous genotype as carriers of hidden abnormalities, is possible only if using proposed DNA technology. Another type of testing breeding animals which is obligatory under law "On Livestock Breeding" is a cytogenetic analysis. In the selection and breeding work it is important not only to use methods for assessing the genetic potential of animals on the basis of productivity traits and availability of genetic diseases caused by mutations in specific genes. We must also take into account the sensitivity of animals’ genetic apparatus to different mutagenic environmental factors; it makes possible to select animals to improve genetic stability of their offspring and creating genetically stable populations at some farms that will significantly reduce the likelihood of chromosomal or genetic reconstructions in animals. Reliable ways to assess genetic and population situation, genetic differences at interspecies, within species and individual level include a method of ISSR-PCR. Effective criteria for determining the impact of mutagenic environmental factors and selection factors on the structure of the investigated samples are implemented via this method and it is used as a tool for search of genetic loci associated with expression of the desired quantitative trait. This development is intended for use primarily in pig breeding for obtaining heterosis of offspring with high potential fattening and meat qualities. As a result of long-term molecular genetic monitoring with use of own-created informative DNA markers in technology ISSR, RAPD, the confirmation of selection achievements in beekeeping was received– creating Khmelnytsky intra-breed type of Ukrainian Steppe bees with construction of unique genetic passport. The basic genetic criteria of "pure breed" of three bee species – Ukrainian, Carpathian and Grey Caucasian Mountain were determined, thus the degree of "pure breed"of bees can be determined accurately on the basis of morpho-metric and molecular genetic indicators. Using ISSR-S4 primer is able to identify the genome fragment of Ukrainian bees with size 950 bp, which is significantly associated with the expression of honey productivity; the research is carried out in this field. Conducting DNA certification of bees to determine the purebred Ukrainian Steppe bees and Carpathian bees is a necessary part of the breeding work in beekeeping for the prevention and elimination of consequences of undesirable interbreed crossbreeding, linear consolidation, increase of traits of honey productivity. In 2014,"Guidelines for morphological and genetic evaluation of Ukrainian bees" were formed and approved by the Ministry of Agrarian Policy of Ukraine as a result of long-term comprehensive studies on the genetics of honey bees. Despite the difficult economic situation, a significant reduction in funding scientific support, reduction of scientific staff, low technological level of existing equipment, the work towards the development of effective technologies for the needs of the agricultural sector is conducted. The unique researches of peculiarities of genetic structure of major histocompatibility complex of cattle and pigs are carried out to identify animals with the best heredity for reproductive capacity and resistance to infectious diseases. Further study of protective and hygienic behaviour of bees, which is currently underway, will enable to improve breeding work in beekeeping with getting environmentally friendly products which can be exported and receive significant foreign exchange earnings to the state budget. In general, livestock of Ukraine is the most important part of the socio-economic development and food security, as it is a manufacturer of biologically important products in the human diet. It is decidedly, that the progressive development of agriculture which defines export potential, quality of life and prosperity of citizens of Ukraine is impossible without the development of modern technologies and appropriate scientific support.
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